Genotoxicity assessment of NIM-76 and its formulation (pessary) in an in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Abstract

The possible genotoxic potential of NIM-76, a volatile fraction obtained from neem oil, having promising contraceptive activity, as well as its formulation product, called pessary (7.5% NIM-76 in polyethylene glycol), were evaluated in the Ames assay and mouse bone marrow micronucleus (MN) assay. Genotoxicity of NIM-76 (0.1–1000?µg/plate) and pessary (0.1–10?000?µg/plate) were studied using the liquid preincubation protocol of the Ames assay both in the presence and absence of S9. Likewise, the ability of NIM-76 [1–1000?mg/kg body weight (b.w.)] and its formulation product (18.75–300?mg/kg b.w.) to induce clastogenic effects were studied in the female mouse bone marrow MN test by using a two-dose intraperitoneal treatment protocol. There was no increase in the number of revertant colonies resulting from NIM-76 or pessary at any of their doses over the respective negative control plates, either in the presence or absence of S9. Similarly, in the MN assay, neither of them showed any clastogenic activity because there was no significant increase in the frequency of micronucleated polychromatic erythrocytes, over the negative control group of animals. The use of this compound in humans is therefore not likely to have mutagenic effects and may be considered as safe with regard to genotoxic potential.     

Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay

Titanium dioxide nanoparticles (TiO₂‐NPs) are being used increasingly for various industrial and consumer products, including cosmetics and sunscreens because of their photoactive properties. Therefore, the toxicity of TiO₂‐NPs needs to be thoroughly understood. In the present study, the genotoxicity of 10nm uncoated sphere TiO₂‐NPs with an anatase crystalline structure, which has been well characterized in a previous study, was assessed using the Salmonella reverse mutation assay (Ames test) and the single‐cell gel electrophoresis (Comet) assay. For the Ames test, Salmonella strains TA102, TA100, TA1537, TA98 and TA1535 were preincubated with eight different concentrations of the TiO₂‐NPs for 4 h at 37 °C, ranging from 0 to 4915.2 µg per plate. No mutation induction was found. Analyses with transmission electron microscopy (TEM) and energy‐dispersive X‐ray spectroscopy (EDS) showed that the TiO₂‐NPs were not able to enter the bacterial cell. For the Comet assay, TK6 cells were treated with 0–200 µg ml^–1 TiO₂‐NPs for 24 h at 37 °C to detect DNA damage. Although the TK6 cells did take up TiO₂‐NPs, no significant induction of DNA breakage or oxidative DNA damage was observed in the treated cells using the standard alkaline Comet assay and the endonuclease III (EndoIII) and human 8‐hydroxyguanine DNA‐glycosylase (hOGG1)‐modified Comet assay, respectively. These results suggest that TiO₂‐NPs are not genotoxic under the conditions of the Ames test and Comet assay. Published 2012. This article is a US Government work and is in the public domain in the USA.     

微核试验和彗星试验检测雄黄的遗传毒性

目的用短期给药(小鼠骨髓细胞微核试验和彗星试验)和长期给药(利用生殖毒性Ⅰ段试验的大鼠做微核试验和彗星试验)检测雄黄的遗传毒性,探讨利用长期毒性试验或生殖毒性Ⅰ段试验用动物进行遗传毒性检测的可行性。方法小鼠ig给予雄黄0.25,0.5和1.0 g·kg-1,每天1次,2 d后,取骨髓细胞做微核试验和彗星试验;利用生殖毒性Ⅰ段大鼠ig给予0.125,0.25和0.55 g·kg-1,雄性连续给药42 d以上,交配成功后处死;雌性连续ig给药19 d以上,妊娠第15天,取骨髓细胞做微核试验和彗星试验,取血做外周血淋巴细胞微核试验。结果与阴性对照组比较,小鼠雄黄0.25,0.5和1.0 g·kg-1组微核试验微核率分别为3.0‰,4.40‰,7.01‰(P<0.05,P<0.01)和彗星试验拖尾率分别为6.3%,9.7%和11.3%(P<0.05,P<0.01)。与阴性对照组比较,生殖毒性Ⅰ段试验大鼠ig给予雄黄,雄黄0.55 g·kg-1组雄性大鼠的骨髓微核和外周血淋巴细胞微核率分别为2.83‰和6.67‰(P<0.05),雌性大鼠0.25和0.55 g·kg-1的骨髓微核和外周血淋巴细胞微核率分别为1.5‰,2.25‰以及2.58‰和4.40‰(P<0.05,P<0.01);雄黄使雄性和雌性大鼠彗星拖尾率明显升高(P<0.05)。结论利用生殖毒性Ⅰ段试验多次给药后取材做微核试验和彗星试验方法可行;外周血微核试验简便易行;在所观察的剂量下雄黄具有遗传毒性。     

Mutagenicity and clastogenicity evaluation of tacrine by Ames and micronucleus assays

Tacrine was evaluated for its mutagenic and clastogenic activities using the Ames bacterial reverse-mutation assay and the rodent bone marrow micronucleus assay. Tacrine was tested for mutagenic potential at six different concentrations, with 1,250 µg/plate as the highest concentration, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, tacrine was administered orally to Wistar rats for 2 days at 5, 10, and 20?mg/kg body weights to assess micronucleus induction in bone marrow erythrocytes. In the Ames assay, tacrine showed nonmutagenicity in four tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, and TA1535, but it was found to be mutagenic in the TA1537 tester strain, both in the presence and absence of a metabolic activation system. Tacrine was found to be nonclastogenic on bone marrow cells of rats at all doses tested and was found to be mutagenic in only the TA1537 strain of Salmonella.     

泰格列净Ames试验研究

目的 应用Ames试验探讨泰格列净是否具有致突变性。方法 采用标准平板掺入法,应用组氨酸营养缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102和TA1535对泰格列净进行回复突变试验。结果 在31.25～2 000μg/皿剂量范围内,加和不加大鼠肝微粒体酶S9平行条件下测试,泰格列净Ames回复突变试验结果为阴性。结论 在本试验条件下,泰格列净无致突变性。     

大鼠肝彗星试验与骨髓微核试验评价结果比较研究

目的 梳理国家药物安全评价监测中心联合开展的大鼠多终点体内遗传毒性试验数据,比较大鼠肝彗星试验与骨髓微核试验结果的一致性和灵敏性。方法 试验分设阴性物质组、作用机制明确的遗传毒性阳性物质组、受试物组,阴性物质包括超纯水、0.9%氯化钠注射液、玉米油、0.5%羧甲基纤维素钠（CMC-Na）、5%蔗糖和聚山梨酯80,给药体积为10 mL·kg^-1;遗传毒性阳性物质包括200 mg·kg^-1甲磺酸乙酯（EMS）、40 mg·kg^-1N-乙基-N-亚硝基脲（ENU）、40 mg·kg^-1环磷酰胺、75 mg·kg^-1甲基苄肼、800 mg·kg^-1尿烷、75 mg·kg^-1对氯苯胺、40 mg·kg^-1 1,2-二溴-3-氯丙烷和10 mg·kg^-1秋水仙素;受试物包括100、300、1000 mg·kg^-1大黄素-8-O-β-D-葡萄糖苷,6.5、65.0、650.0 mg·kg^-1单蒽酮和6.5、65.0、650.0 mg·kg^-1大黄素甲醚。分别在实验0、24、45 hig给药1次,给药体积为10 mL·kg^-1。开展大鼠肝彗星试验和骨髓微核试验,计算每只动物的肝细胞刺猬细胞率和尾DNA百分含量（Tail% DNA）,以及每只动物的嗜多染红细胞（PCE）/总红细胞（ERY）比例和嗜多染红细胞微核（MNPCE）率。结果 大鼠肝彗星试验可有效检出DNA断裂剂,对多种烷化剂（甲磺酸乙酯、甲基苄肼和尿烷等）有较好的预测性,但对环磷酰胺和多倍体诱导剂不灵敏。大黄素-8-O-β-D-葡萄糖苷、单蒽酮和大黄素甲醚骨髓微核试验结果均为阴性。大黄素-8-O-β-D-葡萄糖苷在1 000 mg·kg^-1剂量下导致的肝Tail% DNA与0.5% CMC-Na组比较显著升高（P<0.05）;单蒽酮的肝彗星试验结果为明确阳性,剂量为650 mg·kg^-1时,单蒽酮可导致大鼠肝Tail% DNA显著升高（P<0.05）,且作用存在剂量相关性;大黄素甲醚的肝彗星试验结果为阴性。结论 大鼠肝彗星试验可与骨髓微核试验互补,有效检出主要作用于肝脏且亲电子性较强的遗传毒性化合物。     

Assessment of cytotoxic and genotoxic potential of pyracarbolid by Allium test and micronucleus assay

The present study evaluates the cytotoxic and genotoxic potential of pyracarbolid using both micronuleus (MN) assay, in human lymphocytes, and Allium cepa assay, in the root meristem cells. In Allium test, EC₅₀ value was determined in order to selecting the test concentrations for the assay and the root tips were treated with 25?ppm (EC₅₀/2), 50?ppm (EC₅₀) and 100?ppm (EC₅₀?×?2) concentrations of pyracarbolid. One percent of dimethyl sulphoxide (DMSO) and methyl methane sulfonate (MMS) were used as negative and positive controls, respectively. In the micronucleus assay, the cultures were treated with four concentrations (250, 500, 750 and 1000?µg/ml) of pyracarbolid for 24 and 48?h, negative and positive controls were also used in the experiment parallely. The results showed that mitotic index (MI) significantly reduced with increasing the pyracarbolid concentration at each exposure time. It was also obtained that prophase and metaphase index decreased significantly in all concentration at each exposure time. Anaphase index decreased as well and results were found to be statistically significant, except 24?h. A significant increase was observed in MN frequency in all concentrations and both treatment periods when compared with the controls. Pyracarbolid also caused a significant reduction in the cytokinesis block proliferation index (CBPI) in all concentration and both exposure time.     

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes

Doxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 µg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 µg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.     

Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test

Captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) is a fungicide used to inhibit the growth of many types of fungi on plants used as foodstuffs. The toxic and genotoxic potentials of captan were evaluated with the micronucleus test (MNT; AFNOR,2000) and the comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that captan was toxic (1) to Xenopus larvae exposed to from 2 mg/L to 125 or 62.5 microg/L, depending on the nature of the water [reconstituted water containing mineral salts or mineral water (MW; Volvic, Danone, France)] and (2) to Pleurodeles exposed to from 2 mg/L to 125 microg/L in both types of water. The MNT results obtained in MW showed that captan (62.5 microg/L) was genotoxic to Xenopus but not genotoxic to Pleurodeles at all concentrations tested. CA established that the genotoxicity of captan to Xenopus and Pleurodeles larvae depended on the concentration, the exposure times, and the comet parameters (tail DNA, TEM, OTM, and TL). The CA and MNT results were compared for their ability to detect DNA damage at the concentrations of captan and the exposure times applied. CA showed captan to be genotoxic from the first day of exposure. In amphibians, CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by captan.     

Micronucleus test and comet assay for the evaluation of zebrafish genomic damage induced by erythromycin and lincomycin

An enormous quantity of pharmacologically active principles are currently being introduced into the environment, with consequent escalation of environmental problems, but only a small number of studies are focusing on an assessment of their genotoxic effects. The aim of this article is to assess the genotoxic effects of erythromycin, lincomycin, and of a combination of these two antibiotics on the genome of the zebrafish. The genotoxicity of the two antibiotics was assessed by applying the micronucleus test to erythrocytes and performing a Comet assay on erythrocytes and hepatocytes. The fish were exposed to antibiotics at different concentrations and times of exposure, under standard laboratory conditions. Depending on the different experimental conditions, erythromycin and lincomycin induced a significant increase in DNA migration (tail moment) and a significant increase in micronuleus frequency. We also conducted an analysis on the activation of repair mechanisms when the genotoxic agent was removed. Only a few of the cells displayed a decrease in damage under these test conditions.     

Comparative evaluation of the toxicity and genotoxicity of cadmium in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the comet assay and the micronucleus test

The toxic and genotoxic potential of Cadmium (CdCl(2)) were evaluated by the micronucleus test (MNT) and comet assay (CA) using amphibian larvae (Xenopus laevis and Pleurodeles waltl). Acute toxicity results showed that Cd is toxic to Xenopus larvae exposed from 2 to 50 mg/L and to Pleurodeles from 5 to 50 mg/L, depending on the nature of the water (reconstituted water containing mineral salts or mineral water MW (Volvic)). The MNT results obtained in MW showed that Cd (2 mg/L) is genotoxic to Xenopus, whereas it was not genotoxic to Pleurodeles at all concentrations tested. The CA established that the genotoxicity of Cd to Xenopus and Pleurodeles larvae depends on the concentration, the exposure times, and the comet parameters (Tail DNA, ETM, OTM, and TL). The CA and MNT results were compared for their ability to detect genotoxic effects, considering the concentrations of Cd applied and the exposure time. The CA showed Cd to be genotoxic from the first day of exposure. In amphibians, the CA appears to be a sensitive and suitable method for detecting genotoxicity such as that caused by Cd.     

雷公藤内酯醇的遗传毒性评价

目的 观察雷公藤的主要有效成分之一雷公藤内酯醇的遗传毒性。方法 采用鼠伤寒沙门氏菌回复突变试验(Ames试验)、体外培养CHO细胞染色体畸变试验和小鼠骨髓微核试验检测雷公藤内酯醇的遗传毒性。结果 Ames试验提示在每皿1.6~1000 μg受试剂量下,在加或不加S9代谢活化系统时,受试物对组氨酸缺陷型鼠伤寒沙门氏菌TA97、TA98、TA100、TA102及TA1535所诱发的回复突变菌落数均与溶媒对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示0.01、0.02和0.04 μg/ml 3个剂量的受试物,对加与不加S9代谢活化系统培养的CHO细胞的染色体畸变率无明显影响。小鼠骨髓微核试验设180、360、720 μg/kg 3个剂量,在720 μg/kg剂量时,雷公藤内酯醇有诱发骨髓嗜多染红细胞微核率增高的效应。结论 在本实验条件下,雷公藤内酯醇对鼠伤寒沙门氏菌无致突变性,对哺乳动物培养细胞染色体无致畸变作用,720 μg/kg剂量下对ICR小鼠有诱发骨髓嗜多染红细胞微核率增高的效应。提示雷公藤内酯醇对人体可能具有潜在的遗传毒性。     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

Statistical evaluation of the in vivo micronucleus assay

The statistical evaluation of the in vivo micronucleus assay is focused on multiple contrast tests for comparisons versus the negative control for count data taking the between-animals variability into account. For a possible claim the compound is not genotoxic in the micronucleus assay a proof of safety approach is proposed. For these statistical approaches user-friendly software is free available.     

Mutagenicity and clastogenicity evaluation of metaphenoxy benzyl chloride by ames and micronucleus assays

Metaphenoxy Benzyl Chloride (3-PBC; CAS # 53874-66-1) is a commonly used intermediate in organic synthesis and is a widely used chemical building block. There is likelihood of human contact with 3-PBC in the process of organic or pesticide synthesis and even through as impurity in the product. Further, this chemical has been reported to be found in water reservoirs causing a threat to aqueous flaura and fauna. Genotoxic alert has been suspected for the chemical 3-PBC based on its structure hence, the impetus of present work was to assess the mutagenic potential of 3-PBC using Ames bacterial reverse-mutation assay and in vitro micronucleus assay. 3-PBC was tested for mutagenic potential at six different concentrations, with 0.05 (without metabolic activation) and 0.50 (with metabolic activation) µL/plate as the highest concentrations, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, 3-PBC was tested at concentrations ranged from 0.31 to 4.88?µL/mL to assess micronucleus induction in mammalian cells viz. Chinese Hamster Ovarian-K1 cells (CHO-K1 cells). In the Ames assay, 3-PBC did not show mutagenicity in all five tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, TA1535 and TA1537 both in the presence and absence of a metabolic activation system and found to be nonclastogenic when evaluated in “CHO-K1 cells”.     

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.     

Sublethal toxicity of esbiothrin relationship with total antioxidant status and in vivo genotoxicity assessment in fish (Cyprinus carpio L., 1758) using the micronucleus test and comet assay

Esbiothrin, synthetic pyrethroid with quick activity against insects, is widely used against household pests and in public health. Despite widespread use, data on ecotoxicity and genotoxic effects are extremely scarce. The aim of the present study is to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronucleus test and comet assay in peripheral blood erythrocytes. Effects of two sublethal exposure concentrations on plasma total antioxidant status (TAS mmol/L), and Hct values were examined. On the basis of the 96 h LC₅₀ data from U.S. EPA ecotox database (32 μg/L) two sublethal exposure concentrations (5 and 10 μg/L) were used together with ethyl methanesulfonate (EMS) (5 mg/L) as positive control. Five fish were used for each dose/duration group (24, 48, and 72 h) under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. Plasma TAS (mmol/L) levels decreased in 24 h; an increase was observed slightly for 48 and obviously for 72 h in both exposure doses. Similarly, hematocrit (Hct) values differed between exposure duration but no significant differences in mean values were found between groups of the same exposure time. The general trend was a rise after 48 h, which decreased afterwards. Our results revealed significant increases in the frequencies of micronuclei and levels of DNA strand breaks and thus demonstrated the genotoxic potential of this pesticide on fish, a nontarget organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28:644–651, 2013.     

Detection of genotoxicity and mutagenicity of chlorthiophos using micronucleus,chromosome aberration,sister chromatid exchange,and Ames tests

Potential mutagenic and genotoxic effects of Chlorthiophos, an organophosphate pesticide, were evaluated using four standard assays. Five different concentrations of the pesticide were tested by an Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9 metabolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100, and TA102 strains, with and without S9 fraction, but were all mutagenic to the TA98 strain without S9. Sister chromatid exchange (SCE), chromosome aberration (CA), and micronucleus (MN) tests were used to investigate the genotoxic effects of Chlorthiophos in human peripheral lymphocytes treated with 25, 50, 100, and 200 µg/mL concentrations of Chlorthiophos for 24 and 48 h. The nuclear division index (NDI), replication index (RI), and mitotic index (MI) were also calculated to determine the cytotoxicity of Chlorthiophos. No increase in SCE frequency was seen for any treatment period or concentration, but Chlorthiophos at 200 µg/mL increased the frequency of CAs. Increases in MN formation were only observed at Chlorthiophos concentrations of 200 µg/mL following 24 and 48 h treatments. Chlorthiophos treatment reduced the MI and NDI significantly, but had no effect on the RI. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 937–945, 2015.     

Genetic toxicity assessment: employing the best science for human safety evaluation part III: the comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection.

Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.     

Genotoxicity studies on HM10760A,recombinant human erythropoietin conjugated to globin fragment

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100?μg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100?μg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600?μg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600?μg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.